Abstract

Cytotoxic T lymphocytes (CTL) kill target cells on the basis of cell‐surface antigen recognition and are important in the
host response to tumors, transplants, and viruses. This unit presents several protocols for generating and measuring CTL activity.
The first describes generating CTL against some of the most commonly used target antigens. Two methods for the quantitation
of CTL activity are described based on the two pathways used bt CTL to kill target cells. In one pathway, they release lytic
granules containing perforin and granzymes, leading to apoptosis and target cell lysis. In a second pathway, they trigger
apoptosis via Fas/Fas ligand interactions. In the chromium‐release assay provided here, labeled antigenic targets are recognized
and lysed, releasing radioactivity into the supernatant. In the JAM test protocol, CTL activity is determined by measuring
degradation of radioactively labeled DNA in target cells that have undergone apoptotic cell death. Rather than measuring release
of radioactivity, the JAM test measures the amount of DNA retained in target cells that are not killed by CTL. Two support
protocols detail the generation of CTL precursors (CTLp) against antigens that require priming in vivo. A second set of support
protocols describe the preparation of both stimulator and target cells for these responses using two representative antigens,
trinitrophenyl and viruses. Finally, two alternate protocols illustrate how to determine total CTLp activity in a population
that might express cytolytic activity. These protocols bypass MHC restriction and the original antigen specificity of CTLp
by polyclonal stimulation of CTLp with mitogens followed by attachment of CTL to target cells and subsequent cytolysis.

CAUTION: Follow standard radiation‐safety procedures when working with [3H]thymidine solution and labeled cells.NOTE: All culture incubations are performed in a humidified 37°C, 5% CO2 incubator unless otherwise specified.

Support Protocol 1: In Vivo Priming of Mice with Minor Histocompatibility Antigens